Flow cytometry is a relatively new and rapidly changing technology and has evolved into a sophisticated analytic tool for rapidly quantitating multiple chemical and physical properties of individual cells or cellular constituents of heterogeneous populations. The value of flow cytometry in biological research can be seen in its ability to reveal information concerning the cell cycle kinetics, DNA ploidy levels, and the quantitation of cell surface antibodies. The sophisticated technical achievements made possible by this instrumentation find clinical applications in cancer cell detection, blood cell counting, performing differential white blood cell counts, drug effectiveness studies including cancer chemotherapy, monitoring leukemia in other solid tumors, as well as uses in immunology and virology. Additional biologic applications are continuously being discovered even as the flow cytometry techniques continue to undergo dramatic improvements.
Typically, cell sorting is accomplished by instruments generally measuring a multitude of parameters. Most conventional cell cytometers employ some type of hydrodynamic focusing whereby the cells to be measured are aligned in a single file fashion within a fluid sheath. The cells are then passed by a detector region which measure either a change in electrical properties of a small aperture as the cell passes therethrough or the light scattering effects occasioned by the passage of the cell past a light source. Since different cells typically exhibit varying characteristics, detection of the dynamic effects occasioned by the passage of the cell provides data useful in discriminating and quantitating between cell populations. With the addition of cell sorting capabilities, such an instrument can analyze a mixed population of cells, discriminate between cell types and physically manipulate the cells, generally by employing electrostatic principles or variations thereof, so that they may be concentrated to greater purity. This is generally accomplished by the deposition of the cells into vials or similar type of containers.
In order to monitor the type of cells collected in this fashion and to appropriately adjust the window parameters for the effective discrimination for and collection of desired cell types, the cells must be examined microscopically. With large numbers of cells, this may be accomplished by the deposition of the cells onto a microscope slide, and after staining procedures, easily found because of their pervasive presence on the slide. Great difficulty is encountered, however, when the population of selected cells is very small, i.e. on the order of a hundred cells. Indiscriminate application of such a small number of cells to a microscope slide will force the microscopist on a hunt and search detail in order to assure that he has correctly identified representative cell types in that small population.
In order to reduce this difficult and demanding task, conventional methods have dictated the use of microscope slides having concave areas for the deposition of cells in one spot. This method has been effectively employed in tissue culture and tissue typing since the accumulation of cells in a localized spot has aided in the detection of seralogical reactions. For examination of single cells, this method is not desirable since all the cells are coalesced in a very localized area and consequently, often physically overlap one another. Further, such a system is primarily useful for cells in suspension and does not allow the facile employment of typical staining methods such as Wright stain and the like.
It is an object of the present invention to provide an apparatus capable of permitting the examination of a small number of cells that have been sorted by a cell sorter and to provide their attachment to a microscope slide in a manner consistent with standard staining techniques.
Another conventional method employs a cup for the collection and retention of cells in conjunction with the use of a centrifuge. This system has also proven to be ineffective since the cups are designed to contain a large volume and the desired rare cells are typically in very small numbers. With such a high dilution, it consequently becomes extraordinarily difficult to locate the cells for subsequent staining and examination.
It is an object of the present invention to provide apparatus and methodology whereby rare sorted cells may be permanently attached onto a slide for subsequent staining and examination and that despite the small number of cells, their fixation, staining, and localization in a specified limited site provides for reduced probability of loss and destruction as well as increased handling ease.